Protein tyrosine kinases are essential mediators of axon and dendrite guidance cues. In most cases, the protein targets that mediate the downstream actions of these kinases remain to be identified. The goal of our research is to adapt a biochemical technique, the "bump-hole" approach, to identify tyrosine kinase substrates in the developing brain. The Abl and Arg nonreceptor tyrosine kinase are required for cortical dendrite branch formation in the developing mouse brain. As a proof of concept, we propose to identify and functionally characterize substrates of Abl and Arg in the developing brain. Our first aim is to identify Abl and Arg substrates in developing neurons. We have synthesized a chemically altered form of ATP that contains a benzyl group "bump" on the adenosine ring of ATP (bumped ATP) and engineered a corresponding "hole" in Abl and Arg by removing a bulky amino acid residue from the ATP-binding pocket. Importantly, these altered specificity- (as-) forms of Abl and Arg can utilize the bumped ATP, whereas kinases present in brain extracts cannot. We will selectively label Abl and Arg substrates in vitro by incubating brain extracts with as-Abl or as-Arg in the presence of gamma32P-labeled bumped ATP. We will purify labeled substrates using biochemical fractionation and anti-phosphotyrosine affinity resins and identify them by mass spectrometry. We will use in vitro and cell-based phosphorylation assays to confirm that the identified candidates are bona fide Abl/Arg substrates. Our second aim is to determine whether the substrates regulate axon or dendrite morphogenesis. We will examine the localization of each substrate in cultured wild type and Arg-deficient cortical neurons and determine how this localization is influenced by integrin-mediated adhesion or elevated Arg kinase activity. We will test whether overexpression of the substrate alone or in combination with Arg affects axon or dendrite branching in cultured cortical neurons. We will determine how RNAi-mediated reduction of the regulator influences axon and dendrite branching following adhesion or Arg overexpression. These studies should prove the utility of using the bump-hole approach to identify tyrosine kinase targets in developing neurons. Identifying these substrate will expand the "toolkit" available to dissect the molecular mechanisms of axon and dendrite morphogenesis. [unreadable] [unreadable]